Phenanthridine derivatives and antitumoral medicaments containing phenanthridine

ABSTRACT

The invention relates to novel phenanthridine derivatives and to medicaments, which are for use in antitumoral therapy and prophylaxis and which contain these phenanthridine derivatives. It could be shown that up to now specifically substituted phenanthridine derivatives of the prior art neither have a known nor an antitumoral activity that is to be expected.

[0001] The invention relates to new phenanthridine derivatives as wellas pharmaceuticals for a antitumoural therapy and prophylaxis whichcontain these phenanthridine derivatives. As state of the art WO97/14683 should be mentioned where phenanthridine derivatives andmethods of their preparation are described.

[0002] While the synthesis of phenanthridine derivatives is well knownfor a long time from the technical state and many different syntheticroutes exist, the pharmalogical activity has been detected justrecently. Thus WO 97/14683 describes for the first time the antitumoralactivity of these compounds. However, the antitumoral activity of thedescribed derivatives is limited for a medical application. Startingfrom this, the objective of this invention is to present newphenanthridine derivatives, similar to the ones described in WO97/14683, which show a higher antitumoral activity, in comparison to thestate of the art.

[0003] The objective is achieved with respect to the phenanthridinederivatives by characterising features of claim 1, and with respect topharmaceuticals by the features of claim 7.

[0004] The subclaims demonstrate advantageous further developments. Theapplication of the phenanthridines is mentioned in claims 12 to 16.

[0005] In accordance with the invention new phenanthridine derivativeswith excellent antitumoural and cytotoxic properties could be delivered.They are defined by the general formula I

[0006] The residues R₁ to R₄ are selected in a way that three of themare methoxy groups and one is a hydrogen.

[0007] Surprisingly it could be demonstrated that phenanthridinederivatives substituted in this way have—from the state of theart—unknown and unexpected antitumour properties. This refers to thesubcutaneous as well as to the intraperitoneal application. The knownderivatives from WO 97/14683 show less activity in terms of subcutaneousand intraperitoneal usage.

[0008] The phenanthridine derivatives presented here show excellentanti-tumour, anti-microbial, anti-fungicidal, anti-viral andanti-inflammatory properties. For the studies of the pharmacologicalproperties, the derivatives have been tested in a“in-vivo-antitumor-Screening” of the National Cancer Institute (NCI)Bethesda, Md., USA.

[0009] The derivatives have been tested against a standard panel oftwelve human pathogenic tumor cell lines of six different cancer types(non-small cell lung cancer, melanoma, breast cancer, ovarian cancer,colon cancer, CNS cancer).

[0010] In the preferred way, the derivatives are present in an ionicfrom, especially preferred as salts. In accordance with the inventionthe phenanthridine derivatives exist in acceptable physiologicals salts.Such salts are e.g. salts of inorganic an organic acids, for examplehydrochlorides, hydrobromides and sulfates. Especially well suited saltsfrom organic acids are formed with aliphatic mono- and dicarboxylicacids. Examples are acetates, maleates and fumarates.

[0011] The following derivatives are preferred according to the highestanti-tumour activity:

[0012] A) 6-Amino-11-(3′,4′,5′-trimethoxyphenyl)benzo[c]phenanthridineperchlorate with the structure presented by formula III,

[0013] B) 6-Amino-11,12-dihydro-11-(2′,3′,4′-trimethoxyphenyl)benzo[c]phenanthridine hydrochloride with the structure presented by formula IV

[0014] C) 6-Amino-11,12-dihydro-11-(3′,4′,5′-trimethoxyphenyl)benzo[c]phenanthridine hydrochloride with the structure represented by formulaV

[0015] The invention also relates therefor to medicines containingphenanthridine derivatives which are described here. The medicinecontains, for this purpose, at least one phenanthridine derivative, inthe manner described here, together with at least one inertpharmaceutically acceptable carrier or dilution medium. A derivative ofthe general formula III, IV and/or V is preferred as a phenanthridinederivative. The compound, according to the invention, can beadministered orally, topically or parenterally, or in the form ofsuppositories. The preferred mode of administration is oraladministration. This can be administered in the form of the base or as aphysiologically acceptable salt. It is generally mixed with apharmaceutically acceptable carrier or dilution medium, in order tocreate a medicine. For oral administration the medicine can be madeavailable most usefully in the form of capsules or tablets or possiblyeven slow-release tablets. They can also be available in the form ofdragees or in syrup form. Suitable topic preparations are e.g. salts,lotions, creams, powders and sprays.

[0016] In the same way the invention concerns the usage of at least oneof the above named phenanthridine derivatives for the preparation of amedicine for an anti-tumour therapy and prophylaxis.

[0017] The further examples and figures describe in greater detail theproperties of the compounds presented in this invention.

EXAMPLE 1 Synthesis of6-Amino-11-(3′,4′,5′-trimethoxyphenyl)benzo[c]phenanthridine Perchlorate

[0018] To a solution of 4.85 mmol (2.0 g)6-Amino-11,12-dihydro-11-(3′,4′,5′-tri-methoxyphenyl)benzo[c]phenanthridinein 120 ml dioxane a solution of 19.4 mmol (4.63 g) DDQ in 100 ml dioxaneis added and heated under reflux for 16 h. The cooled reaction mixtureis than hydrolysed with 500 ml saturated sodium hydrocarbonate solutionand stirred vigorously for 30 minutes. The aqueous phase is thanextracted three times with 200 ml ether, the combined organic extractswere washed once with 300 ml saturated sodium hydrocarbonate solutionand three times with 250 ml water. Then the organic phase is dried oversodium sulfate if needed and rotated to about 150 ml. For precipitationof the6-Amino-11,12-dihydro-11-(3′,4′,5′-trimethoxy-phenyl)benzo[c]phenanthridineperchlorate the organic phase is stirred over night with 10 ml 70%perchloric acid or if needed with 20 ml 70% perchloric acid, ethanol andether (1/1/1).

[0019] The brown precipitation is collected, washed with a small portionof ether and recrystallized in methanol and dried for 24 h in an oilpump vacuum.

[0020] Yield: 1.12 g (44%) of theory), darkbrown needles

[0021] melting point: 296° C. C₂₆H₂₃N₂O₇Cl (510.93) Calc.: C 61.12 H4.54 N 5.48 O 21.92 Cl 6.94 Found: C 61.10 H 4.55 N 5.40

[0022]¹H-NMR (DMSO-d₆, 300 MHz): δ/ppm (TMS)=3.73 (s, 6H, 2 —OCH₃), 3.79(s, 3H, —OCH₃), 6.77 (s, 2H, ArH), 7.65-7.95 (m, 6H, ArH), 8.14 (d, 1H,ArH), 8.67 (d, 2H, ArH), 9.82 (br, s, 2H, —NH₂), 12.95 (br, s,1H,≡-N⁺—H).

[0023]¹³C-NMR (DMSO-d₆, 75 MHz): δ/ppm (TMS)=56.1 (2C, 2 —OCH₃), 60.3(—OCH₃), 106.3 (2C), 121.1, 126.1, 127.6, 127.9, 128.1, 128.4, 128.7,128.9, 133.2 (11C, C-1, -2, -3, -4, -7, -8, -9, -10, -12, -2′, -6′),114.9, 118.1, 121.8, 130.4, 132.3, 134.2, 136.6, 137.4, 137.7, 153.5(2C), 154.6, (12C, C-4a, -4b, -6, -6a, -10a, -10b, -11, -12a, -1′, -3′,-4′, -5′).

[0024] MS (EI): m/z (%)=410 (M⁺ of the base, 100), 395 (M⁺—CH₃, 20), 380(M⁺−2CH₃,3), 363 (6), 352 (9), 335 (8), 320 (9), 305 (8), 292 (15), 281(10).

[0025] IR (KBr): v/cm⁻¹=3355, 3234, 1683, 1595, 1517.

EXAMPLE 2 Synthesis of6-Amino-11,12-dihydro-11-(2′,3′,4′-trimethoxyphenyl)benzo[c]-phenanthridinehydrochloride

[0026] A solution of 80 mmol 2-methylbenzonitrile (9.36 g) and 40 mmol2,3,4-tri-methoxybenzaldehyde (7.85 g) in 40 ml DMPU was added dropwiseto a stirred solution of 88 mmol potassium-tert-butylate (9.86 g) in 90ml DMPU at 30° C. under an atmosphere of nitrogen. The resulting mixturewas stirred for 3-4 h at 35-40° C. and then decomposed with 400 ml icewater containing 80 mmol ammonium chloride (8.8 g). The aqueous layerwas extracted three times with 200 ml methylene chloride. Afterevaporation of the solvent, 20 ml 5N hydrochloric acid was added undervigorous stirring to obtain6-amino-11,12-dihydro-11-(2′,3′,4′-trimethoxyphenyl)benzo[c]phenanthridinehydrochloride. The colourless precipitate was collected, washed withmethylene chloride, recrystallized from methanol/water (3:1) and driedin an oil pump vacuum for 24 h. Yield: 11,40 g (64% of theory),colourless crystals melting point: 283° C. C₂₆H₂₅N₂O₃Cl (448.95) Calc.:C 69.56 H 5.61 N 6.24 O 10.69 Cl 7.90 Found: C 69.51 H 5.48 N 6.21

[0027]¹H-NMR (DMSO-d₆, 300 MHz): δ/ppm (TMS)=3.08 (d, 1H, H-12a,²J_((H-12a,H-12b))=15.8 Hz), 3.52 (mc, 1H, H-12b), 3.59 (s, 3H, —OCH₃),3.79 (s, 3H, —OCH₃), 4.13 (s, 3H, —OCH₃), 5.04 (d, 1H, H-11,³J_((H-1,H-12b))=7.0 Hz), 6.04 (d, 1H, ArH), 6.31 (d, 1H, ArH), 7.23 (d,1H, ArH), 7.35 (mc, 1H, ArH), 7.44 (mc, 1H, ArH), 7.73 (d, 2H, ArH),7.93 (mc, 1H, ArH), 8.48 (d, 1H, ArH), 8,62 (d, 1H, ArH), 9.80 (br, s,2H, —NH₂), 14.04 (br, s, 1H, ≡N⁺—H).

[0028]¹³C-NMR (DMSO-d₆, 75 MHz): δ/ppm (TMS)=30.2 (C-11), 34.8 (C-12),55.5 (—OCH₃), 60.3 (—OCH₃), 61.2 (—OCH₃), 107.1, 123.4, 123.8, 125.8,126.2, 127.1, 128.1, 129.2, 130.1, 135.0 (10C, C-1, -2, -3, -4, -7, -8,-9, -10, -5′, -6′), 117.4, 117.5, 121.8 133.0, 135.2, 135.7, 141.7,150.3, 152.3, 155.3 (11C, C-4a, -4b, -6, -6a, -10a, -10b, -12a, -1′,-2′, -3′, -4′).

[0029] IR (KBr): v/cm⁻¹=3258, 3072, 2934, 2834, 1642, 1620, 1600 ,1570,1554.

[0030] Release of the base:

[0031] A solution of6-Amino-11,12-dihydro-11-(2′,3′,4′-trimethoxy-phenyl)benzo[c]phenanthridinehydrochloride in 20 ml conc. ammonia and 250 ml diethyl ether wasstirred vigorously for 1 h. The organic layer was separated and pouredinto 350 ml petroleum ether (50-70). The precipitate was collected.After evaporation of the solvent, further precipitate was collected andwashed three times with a small amount of petroleum ether. Thecolourless crystals of6-Amino-11,12-dihydro-11-(2′,3′,4′-trimethoxy-phenyl)benzo[c]phenanthridinewere dried in an oil pump vacuum for 24 h. melting point: 137° C.C₂₆H₂₄N₂O₃ (412.49) Calc.: C 75.71 H 5.86 N 6.79 O 11.64 Found: C 75.65H 5.88 N 6.65

[0032]¹H-NMR (DMSO-d₆, 300 MHz): δ/ppm (TMS)=3.04 (d, 1H, H-12a,²J_((H-12a,H-12b))=15.5 Hz), 3.57 (mc, 1H, H-12b), 3.63 (s, 3H, —OCH₃),3.89 (s, 3H, —OCH₃), 4.18 (s, 3H, —OCH₃), 5.15 (d, 1H, H-11,³J_((H-11,H-12b))=7.3 Hz), 5.20 (br, s, 2H, —NH₂), 6.12 (mc, 2H, ArH),7.04 (d, 1H, ArH), 7.19 (mc, 1H, ArH), 7.31-7.41 (m, 2H, ArH), 7.54 (mc,1H, ArH), 7.80 (mc, 2H, ArH), 8.36 (d, 1H, ArH).

[0033]¹³C-NMR (DMSO-d₆, 75 MHz): δ/ppm (TMS)=31.3 (C-11), 39.9 (C-12),55.6 (—OCH₃), 60.7 (—OCH₃), 61.3 (—OCH₃), 106.8, 122.9, 123.1, 123.7,124.5, 125.4, 126.8, 128.2, 130.4 (10C, C-1, -2, -3, -4, -7, -8, -9,-10, -5′, -6′), 118.5, 119.3, 129.3, 135.7, 135.9, 136.9, 142.5, 144.9,151.2, 152.7, 156.0 (11C, C-4a, -4b, -6, -6a, -10a, -10b, -12, -1′,-2′,-3′, -4′).

[0034] MS (EI): m/z (%)=412 (M⁺, 95), 397 (M⁺−CH₃, 0), 381 (M⁺ 13 OCH₃,37), 245 (M⁺−2,3,4-Trimethoxyphenyl, 100), 231 (59), 201 (10), 168 (5),153 (7).

[0035] IR (KBr): v/cm⁻¹=3426, 3310, 3178, 2918, 2815, 1630, 1595, 1557.

EXAMPLE 3 Synthesis of6-Amino-11,12-dihydro-11-(3′,4′,5′-Trimethoxyphenyl)benzo[c]-phenanthridineHydrochloride

[0036] A solution of 80 mmol 2-methylbenzonitrile (9.36 g) and 40 mmol3,4,5-tri-methoxybenzaldehyde (7.85 g) in 40 ml DMPU was added dropwiseto a stirred solution of 88 mmol potassium-tert-butylate (9.86 g) in 90ml DMPU at 30° C. under an atmosphere of nitrogen. The resulting mixturewas stirred for 3-4 h at 35-40° C. and then decomposed with 400 ml icewater containing 80 mmol ammonium chloride (8.8 g). The aqueous layerwas extracted two times with 250 ml methylene chloride. Afterevaporation of the solvent, 10 ml conc. hydrochloric acid was addedunder vigorous stirring to obtain6-amino-11,12-dihydro-11-(3′,4′,5′-trimethoxyphenyl)benzo[c]phenanthridinehydrochloride. The pale precipitate was collected, washed with methylenechloride, recrystallized from methanol/water (3:1) and dried in an oilpump vacuum for 24 h. Yield: 10.7 g (60% of theory), colourless crystalsmelting point: 272° C. C₂₆H₂₅N₂O₃Cl (448.95) Calc.: C 69.56 H 5.61 N6.24 O 10.69 Cl 7.90 Found: C 69.48 H 5.53 N 6.17

[0037]¹H-NMR (DSMO-d₆, 300 MHz): δ/ppm (TMS)=3.13 (d, 1H, H-12a,²J_((H-12a,H-12b))=15.7 Hz), 3.50 (s, 6H, 2 —OCH₃), 3.5* (mc, 1H,1H-12b), 3.52 (s, 3H, —OCH₃), 4.83 (d, 1H, H-11, ³J_((H-11, H-12b))=6.5Hz), 6.34 (s, 1H, ArH), 7.27 (d, 1H, ArH), 7.38 (mc, 1H, ArH), 7.45 (mc,1H, ArH), 7.75 (mc, 1H, ArH), 7.98 (mc, 1H, ArH), 8.04(d, 1H, ArH), 8.34(d, 1H, ArH), 8.62 (d, 1H, ArH), 9.55 (br, s, ₂H, —NH2), 13.87 (br, s,1H, ≡N⁺—H).

[0038]¹³C-NMR (DMSO-d₆, 75 MHz): δ/ppm (TMS)=35.9 (C-12), 36.7 (C-11),55.6 (2C, 2 —OCH₃), 59.8 (—OCH₃) 104.6 (2C), 123.4, 124.5, 126.1, 127.1,128.0, 129.2, 130.1, 135.1 (10C, C-1, -2, -3, -4, -7, -8, -9, -10, -2′,-6′), 117.5, 117.9, 127.6, 132.7, 135.2, 135.9, 136.2, 137.1, 152.6(2C), 155.3 (11C, C-4a, -4b, -6, -6a, -10a, -10b, -12a, -1′, -3′, -4′,-5′). IR (KBr): v/cm⁻¹=3388, 3156, 2940, 2842, 1665, 1633, 1598.

[0039] Release of the base:

[0040] A solution of6-Amino-11,12-dihydro-11-(3′,4′,5′-trimethoxy-phenyl)benzo[c]-phenanthridinehydrochloride in 10 ml conc. ammonia and 100 ml diethyl ether wasstirred vigorously for 1 h. The organic layer was separated and pouredinto 150 ml petroleum ether (50-70). The precipitate was collected.After evaporation of the solvent, further precipitate was collected andwashed two times with a small amount of petroleum ether. The colourlesscrystals of6-Amino-11,12-dihydro-1-(3′,4′,5′-trimethoxyphenyl)-benzo[c]phenanthridinewere dried in an oil pump vacuum for 24 h. melting point: 182° C.C₂₆H₂₄N₂O₃ (412.49) Calc.: C 75.71 H 5.86 N 6.79 O 11.64 Found: C 75.70H 5.81 N 6.68

[0041]¹H-NMR (DMSO-d₆, 300 MHZ): δ/ppm (TMS)=3.07 (d, 1H, H-12a,²J_((H-12a,H-12b))=15.7 Hz), 3.54* (mc, 1H, H-12b), 3.55 (s, 6H, 2—OCH₃), 3.70 (s, 3H, —OCH₃), 4.66 (d, 1H, H-11, ³J_((H-11,H-12b))=6.7Hz), 4.74 (br, s, 2H, —NH₂), 6.25 (s, 2H, ArH), 7.05 (d, 1H, ArH), 7.19(mc, 1H, ArH), 7.30-7.40 (m, 2H, ArH), 7.55 (mc, 1H, ArH), 7.82 (mc, 2H,ArH), 8.33 (d, 1H, ArH).

[0042]¹³C-NMR (DMSO-d₆, 75 MHz): δ/ppm (TMS)=37.8 (C-12), 39.9 (C-1),56.5 (2C, 2 —OCH₃), 61.3 (—OCH₃), 105.3 (2C), 123.8, 124.3, 125.1,126.1, 127.6, 128.9, 129.1, 131.0 (10C, C-1, -2, -3, -4, -7, -8, -9,-10, -2′, -6′), 118.4, 118.9, 135.5, 136.0 (2C), 137.0, 139.7, 144.7,153.5 (2C), 156.1 (11C, C-4a, -4b, -6, -6a, -10a, -10b, -12a, -1′, -3′,-4′,-5′).

[0043] MS(EI): m/z (%)=412 (M⁺, 50), 397 (M⁺—CH₃, 8), 245(M⁺-3,4,5-trimethoxyphenyl, 68), 206 (9), 201 (6), 168 (3), 153 (11), 77(8), 43 (100).

[0044] IR (KBr): v/cm⁻¹=3495, 3382, 2943, 2848, 1632, 1592, 1508.

Pharmacological Test Results of the Compounds of Examples 1 to 3

[0045] In order to examine the pharmacological properties, thesecompounds (BP-7, BP-8, BP-D8) as well as6-Amino-11-(2′,4′-dimethoxyphenyl)benzo[c]-phenanthridine perchlorate(BP-D3) [PCT/WO 97/14683] were examined in an “in vivo-hollow fiberassay” of the National Cancer Institute (NCI), Bethesda, Md., USA.

[0046] The Compounds were tested against a panel of 12 human pathogenictumour cell lines of six types of cancer (non-small cell lung cancer,melanoma, breast cancer, ovarian cancer, colon cancer, CNS cancer).

[0047] These cell lines were cultivated in polyvinylidenfluorine hollowfibers which are permeable to small molecules and which were implantedinto nude mice. Three hollow fibers were implanted every animalintraperitoneally and subcutaneously which means three tumor cell linescould be examined in each animal in two physiological sites.

[0048] The animals were treated four times with a dose of the testsubstance with a time interval of 24 hours. For this purpose, everycompound was administered at two concentrations. At the end of thetreatment the hollow fibers were regained and the number of the vitaltumour cells or rather of the protein biomass was determinedphotometrically by an MTT-Formazan-Assay in order to determine theinhibition of growth by the phenanthridines. Mice with prepaired tumourcell cultures in hollow fibers which were only treated by solvent servedas control.

[0049] The evaluation of test results is based on a point system givenby NCI.

[0050] Each administration of a test substance which leads to areduction of the tumour cell growth of at least 50% is given a score oftwo points. The maximum number of points for one compound is therefore96 points (12 cell lines * 2 implantation sites * 2 concentrations ofthe test substance * 2 points). In addition for further specificationthe results on subcutaneously and intraperitoneally implanted hollowfibers are treated separately. There are three criteria given by NCI. Atest substance in the “in vivo-hollow fiber assay” has to fulfill atleast one criterion in order to be regarded as suitable for furthertests:

[0051] (a) the total sum of the points from intraperitoneal andsubcutaneous application must be higher than 20.

[0052] (b) The total sum of the points of the subcutaneous applicationmust be higher than 8.

[0053] (c) After the treatment the vital tumour cell mass of at leastone cell line must be less than the starting value (cell killcharacteristics).

[0054] Out of all tested phenanthridines the6-Amino-11-(3′,4′,5′-trimethoxyphenyl)-benzo[c]phenanthridineperchlorate (BP-D7) showed the highest activity in the “in vivo-hollowfiber assay” and fulfilled the criteria a, b and c (see tab. 1). TABLE 1Results of the phenanthridines in the in vivo-hollow-fiber assay of theNational Cancer Institute Points Points (intra- Points Cell killingCompound (sum) peritoneal) (subcutaneous) properties BP-D7 28 14 14 YesBP-8 20 10 10 Yes BP-7 16 6 10 Yes BP-D3 10 2 8 Yes

[0055]6-Amino-11,12dihydro-11-(2′,3′,4′-trimethoxyphenyl)benzo[c]-phenanthridinehydrochloride (BP-8) and6-Amino-11,12-dihydro-11-(3′,4′,5′-tri-methoxy-phenyl)benzo[c]phenanthridinehydrochloride (BP-7) fulfill the criteria (b) and (c), on the otherhand, 6-Amino-11-(2′,4′-dimethoxy-phenyl)benzo[c]-phenanthridineperchlorate (BP-D3) fulfills the criteria(c) only. Thus thephenanthridines BP-D7, BP-8, and BP-7 achieve higher points than thecompound BP-D3 in all separate categories (subcutaneous andintraperitoneal) as well as in the total sum of all points.

[0056] The FIG. 1 shows the test results for these phenanthridines in aform of a bar chart.

We claim:
 1. A Phenanthridine derivative of the general formula I

wherein three of the rests R₁ to R₄ are methoxy and one is hydrogen. 2.Phenanthridine derivatives according to claim 1 wherein the derivativesare in ionic form.
 3. Phenanthridine derivatives according to claim 1wherein the derivatives are salts.
 4. Phenanthridine derivativesaccording to least one of the claims 1 to 3 of the formula III.


5. Phenanthridine derivatives according to at least one of the claims 1to 3 of the formula IV.


6. Phenanthridine derivatives according to at least one of the claims 1to 3 of the formula V.


7. A pharmaceutical composition for antitumoural therapy and prophylaxiscontaining at least one of the phenanthridine derivative according toprevious claims and optionally at least one pharmaceutical carrierand/or dilution medium.
 8. A pharmaceutical composition according toclaim 7 containing at least one of the phenanthridine derivatives of theformula III to V.
 9. A pharmaceutical composition according to at leastone of the claims 7 or 8 containing at least one dilution medium.
 10. Apharmaceutical composition according to at least one of the claims 7 to9, where the pharmaceutical composition is a formulation for oral use.11. A pharmaceutical composition according to at least one of the claims7 to 9 where the pharmaceutical composition is a formulation for topicaluse.
 12. Method of treating a tumor or of tumor prophylaxischaracterized by administering an effective dosage of at least one ofthe phenanthridine derivatives according to claims 1 to 6 to a mammalianin need thereof.
 13. Method according to claim 12, wherein thephenanthridine derivative is at least one compound of the formula III toV.
 14. Method according to one of the claims 12 or 13, wherein inaddition pharmaceutical carriers and/or dilution media are used. 15.Method according to one of the claims 12 to 14, wherein thepharmaceutical composition is a formulation for oral use.
 16. Methodaccording to one of the claims 12 to 14, wherein the pharmaceuticalcomposition is a formulation for topical use.